Figure 4

High-throughput gene knockout and validation. (A) Schematic representation of the recombination between the pCC1 or pHHT-TK-RH3KO plasmids and the PfRH3 target locus. Primers used to detect 5' (ko5'-fw and rv), 3' (ko3'-fw and rv) loop-ins and WT ORF (wt-fw and rv) are depicted as small arrows. Restriction fragment lengths are shown to scale. 5' and 3' probes are represented as two thick bars. (B) Whole-cell PCR on single cell clones of putative knockouts. Lanes show products for the 5', 3' loop-ins and WT ORF for each individual clonal isolate. Upper panels, pCC1-Δrh3 clonal isolates derived from two stably transfected culture wells (KO1 and 2). Lower panels, pHHT-TK-Δrh3 clonal isolates derived from six stably transfected culture wells (KO1-6). (C) Southern blot of genomic DNA from 3D7 WT strain, parent W2 WT strain, and both pCC1 KO1 and KO2 generated strains.